Fluorescent dyes are widely used in biological research and medical diagnostics. Fluorescent dyes are superior to conventional radioactive materials because fluorescent dyes are typically sufficiently sensitive to be detected, less expensive and less toxic. In particular, a diversity of fluorophores with a distinguishable color range has made it more practical to perform multiplexed assays capable of detecting multiple biological targets in parallel. The ability to visualize multiple targets in parallel is often required for delineating the spatial and temporal relationships amongst different biological targets in vitro and in vivo. In addition, the generation of a wide range of fluorescent dyes has opened a new avenue for conducting high-throughput and automated assays, thus dramatically reducing the unit cost per assay. Moreover, the low toxicity of fluorescent dyes provides ease of handling in vitro, and also renders it safer for imaging biological activities in vivo.
Despite the various advantages of fluorescent dyes, conventional dyes have a number of profound limitations. For example, conventional fluorescent dyes are typically prone to inter-dye quenching, a phenomenon known to diminish the effective brightness of the dyes. It is a common practice to conjugate a given target with multiple dye molecules in order to maximize the brightness of the labeled target, e.g., a biomolecule such as protein or DNA. For many conventional fluorescent dyes, the fluorescence intensity of the labeled target is often not directly proportional to the number of attached dye molecules, but rather less than the predicted intensity due to, e.g., quenching amongst the multiple dyes attached to the target. Such quenching effect can be attributed to, in part, the physical interaction amongst the attached dye molecules, which may lead to formation of nonfluorescent dye dimers. Dimer formation may be driven by hydrophobic interaction. Because many traditional fluorescent dyes, such as various rhodamine dyes and cyanine dyes, are highly hydrophobic aromatic compounds, these commonly used dyes are particularly prone to forming dimers on labeled biomolecules. Adding sulfonate groups to a dye has been shown to reduce dimer formation. See, e.g., U.S. Pat. Nos. 5,268,486 and 6,977,305, 6,130,101 and Panchuk-Voloshina, et al. J. Histochem. Cytochem. 47(9), 1179 (1999). However, while sulfonation may reduce dimer formation, it also introduces negative charges into a biomolecule, and thus may increase the risk of disrupting the biological activity of the labeled biomolecule.
Sulfonated dyes (e.g., AF488, Alexa Fluor 532, Alexa Fluor 546 and Alexa Fluor 568) are useful for antibody labeling or labeling of other macromolecules where multiple dye molecules are typically in close proximity. In some instances, where reduction of background fluorescence signal relies on dye to dye interaction, the activity of the labeled biomolecule may be affected by the charge of the dye. In some instances, labeled antibodies may produce high background in certain cellular staining. To lower the background, it may be necessary to use a negatively charged polymer to act as a blocking agent (US patent application 2008/0038772).
In order to maximize fluorescence signals, fluorescent dyes need to be excited at or near their absorption maxima. The wavelengths of the existing excitation light sources are limited. For example, a 488 nm argon laser is commonly used in fluorescence microscopy, flow cytometry, PCR instruments, DNA sequencing instruments and other fluorescence-based biomedical instruments. Many conventional dyes are prone to poor photostability and undergo rapid photobleaching under intense laser light, a phenomenon known to diminish the effective brightness of dyes when a fluorescence signal is to be followed over time. For many conventional dyes, the fluorescence is also sensitive to pH changes.
In some instances, sulfonation of dyes overcomes the drawbacks of low photostability and pH sensitivity. Sulfonation also increases the water solubility and blue-shifts the absorption wavelength of a dye to a wavelength close to the 488 nm argon laser line. However, the multiple sulfonate groups make a dye relatively insoluble in nonpolar organic solvents. In certain instances, solubility in nonpolar organic solvents is desirable for labeling reactions involving a relatively nonpolar substrate.